Structure-Activity Relationship of USP5 Inhibitors

Area of Science:

• Biochemistry
• Chemical Biology
• Enzymology

Background:

• Ubiquitin-specific protease 5 (USP5) is a deubiquitinase implicated in various diseases, notably cancer.
• Currently, no specific chemical probes exist to target USP5.
• USP5 possesses a poorly characterized zinc-finger ubiquitin-binding domain (ZnF-UBD) crucial for its function.

Purpose of the Study:

• To develop a chemical probe targeting the USP5 ZnF-UBD.
• To inhibit USP5 catalytic activity through targeted binding.
• To establish a chemical and structural foundation for future USP5 research.

Main Methods:

• Structure-activity relationship (SAR) studies of a chemical series targeting the USP5 ZnF-UBD.
• Crystallographic characterization of the ZnF-UBD in complex with various ligands.
• Biochemical assays to assess binding affinity (K_D) and inhibitory activity.

Main Results:

• Identification of compound 64, which binds the USP5 ZnF-UBD with a K_D of 2.8 μM.
• Compound 64 demonstrated selectivity over nine other proteins with similar ZnF-UBD domains.
• Compound 64 inhibited USP5's catalytic cleavage of di-ubiquitin in vitro.

Conclusions:

• A chemical series was developed that targets the USP5 ZnF-UBD and inhibits enzyme activity.
• Compound 64 represents a selective inhibitor and a valuable starting point for a USP5 chemical probe.
• This work provides a framework for future investigations into USP5's cellular roles.